The present invention is directed to methods of isolation and purification of Streptomyces griseus trypsin (SGT) from PRONASE protease in a single affinity chromatography step and uses of the purified SGT.
Trypsin is a serine protease present in the digestive tract of a wide variety of mammals. Its function is the hydrolytic cleavage of peptide bonds, thus reducing the size of large proteins and making them accessible to further degradation by other proteases. Trypsin is used in biotechnological applications, especially in the cultivation of mammalian cells, where it serves as tool for the disintegration of large cell aggregates, or for the removal of cells from surfaces like microcarriers or cultivation trays. Trypsin is also used as a protein degrading enzyme in the processing of non-trypsin sensitive biopolymers. Because of its well known specificity, trypsin is also used as a selective protein cleavage tool in both analytical and preparative processes. Trypsin can be inactivated or inhibited by a number of specific or non-specific protease inhibitors, many of them belonging to the serpine family. The most widely used in biotechnological applications is a trypsin inhibitor from soy beans. As most of these trypsin inhibitors are very specific, they are inactive against other contaminating proteases.
Trypsin is typically prepared from the duodenal glands of various animal species and purified to different grades of purity. The purification of trypsin can be performed by a number of different biochemical processes, including precipitation, ion exchange chromatography and affinity chromatography. It has been shown that prepurified bovine trypsin (Type I) binds to benzamidine immobilized on an insoluble carrier and can be eluted by high concentrations of guanidine or arginine or by decreasing the pH of the eluant (Ellouali et al. 1991. Chromsymp. 2215:255-265). The mammalian pancreas from which trypsin is derived also contains the serine protease chymotrypsin, which is very similar to trypsin in its physiochemical properties, including the interaction with and affinity to amidine derivatives. As a result, these two proteins are difficult to separate. Depending on the purification method, purified trypsin preparations therefore may contain varying amounts of contaminating enzymes, particularly chymotrypsin. Furthermore, mammalian-derived trypsin may contain adventitious agents, such as viruses and prions. Since the discovery of the action of TSE agents, and the possibility of their transmission to humans, there is an ongoing discussion about the use of human or animal derived materials in biotechnology processes providing pharmaceuticals for human use.
PRONASE protease from the microbial organism Streptomyces griseus (S.g.), is a commercially available alternative to trypsin prepared from animal tissues. PRONASE protease has been used for the preparation of primary cell cultures from tissues and for the detachment of cells from surfaces, microcarrier cell cultures and growth of VERO cells in suspension in serum-free media (Weinstein 1966. Exper. Cell Res. 43:234-236; Manousos et al. 1980. In vitro 16:507-515, Litwin 1992. Cytotechn. 10:169-174). The exact mechanism of its action is not known. PRONASE protease is known to be a mixture of different enzymes, including various types of endopeptidases, (serine and metalloproteases), exopeptidases (carboxypeptidase and aminopeptidase), neutral protease, chymotrypsin, trypsin, carboxypeptidase, aminopeptidase, and neutral and alkaline phosphatase.
After enzyme treatment, the activity of trypsin is usually neutralized by the addition of fetal calf serum, which contains a number of specific and non-specific protease inhibitors. However, media free of serum and protein (particularly from mammalian sources) are preferred in cell culture media used for production of vaccine and therapeutic proteins. Therefore, use of serum-free media, which are devoid of any trypsin inhibitor activity, makes it necessary to identify new sources of inhibitor activity. Because PRONASE protease is a mixture of a variety of proteases, inhibition of protease activity requires a mixture of different inhibitors, leading to a very complex and expensive process. The protein load arising from use of PRONASE protease and the composition of inhibitors in a serum-free culture therefore would be much higher compared to a culture using mammalian-derived trypsin and specific trypsin-inhibitor. Furthermore, the addition of PRONASE protease to the culture medium would also adversely effect the purification process, because more protein is present in the medium.
The trypsin-like activity of PRONASE protease, commonly known as Streptomyces griseus trypsin (SGT) shows a sequence identity of approximately 33% to bovine trypsin (Olafson et al. 1975. Biochem 14:1168-1177). Strepomyces griseus trypsin has been purified by chromatographic techniques using different types of ion exchange resins. These methods typically use stable matrices, which minimise the problem of bleeding of the ligand into the product during elution. These methods, however, have relatively low selectivity, leading to purification factors in the range of  less than 10. As a result, to achieve a high degree of purity, several steps have to be combined, which in turn may cause autodigestion of the trypsin and therefore loss of activity. Purification by ion-exchange chromatography on CM-Sephadex, with further purification performed by rechromatography on an ion exchange column has been described by Jurasek et al. (1971. Can. J. Biochem. 49:1195-1201) and Olafson et al. (1975a. Biochem. 14:1168-1177; 1975b, Biochem. 14: 1161-1167). Miyata et al. (1991. Cell Structure and Function 16:39-43) describe a three step cation exchange chromatography process to purify SGT. SGT is found to migrate as a single band in PAGE with a molecular weight of about 30,000 and having an esterase activity higher than bovine trypsin as determined by BAEE assay. However, even SGT purified by three step chromatography purification methods was found to be slightly contaminated by carboxypeptidase B-like activity.
SGT has also been purified from PRONASE protease by affinity chromatography using oligopeptides derived from tryptic digest of salmine as highly specific ligand for SGT. Elution of the trypsin-like activity from the mixture of protease in PRONASE protease with HCl revealed purified SGT which was, however, found to be contaminated by carboxypeptidase B-like activity (Kasei et al. 1975. J. Biochem. 78.:653-662; Yokosawa et al. 1976. J. Biochem. 79:757-763). For analytical purposes only, SGT was also separated from PRONASE protease by affinophoresis using benzamidine as a ligand (Shimura et al. 1982. J. Biochem. 92:1615-1622).
There exists a need for a simple large-scale method for isolation and separation of the active trypsin-like fraction of PRONASE protease. This would allow a controlled system for use in cell culture methods and provide a defined activity of the fraction from a microbial source, which would not bear the risk of contaminants of human pathogens.
There also exists a need to avoid contaminants derived from cell culture medium additives during cell propagation/growth, biomass production and product production process. Reduction of the protein load in a cell culture medium would allow production of highly pure protein products using conventional purification methods.
It is therefore an object of the present invention to provide for a method for isolation of purified Streptomyces griseus trypsin (SGT) from PRONASE protease.
It is also an object of the invention to provide for a preparation of purified SGT having high specific activity.
It is another object of the invention to provide for use of purified SGT in biotechnology processes.
It is another object of the invention to provide for use of using purified SGT for production of a biomass of eukaryotic cells.
It is another object of the invention to provide for use of purified SGT for production of virus or virus antigen.
It is also an object of the invention to provide for use of purified SGT for production of biological products with cells passaged and subcultured with purified SGT only.